Amplicon Sequencing (16S) vs. Shotgun Sequencing (Metagenomics)

Marker-Gene (Amplicon) Sequencing vs Whole-Genome Shotgun

• Amplicon sequencing targets a specific genetic marker (e.g. the 16S rRNA gene for bacteria, ITS for fungi) and amplifies it by PCR from the community DNA. This produces sequencing reads for the same locus across different organisms, enabling identification of taxa present. It is also called marker gene sequencing or metabarcoding.
• Shotgun metagenomic sequencing directly sequences all DNA extracted from a sample, without PCR bias. Random fragments of all genomes in the community are sequenced (“shotgun” approach), capturing the whole genome of microorganisms (and viruses) present.
• Data output differs: Amplicon sequencing yields a list of taxa and their relative abundances – essentially a community profile (which organisms are present). In contrast, shotgun metagenomics yields sequences from all genes, allowing assembly of genomes and analysis of functional genes and pathways present in the community.
• Example: 16S rRNA gene amplicon sequencing was foundational in microbial ecology – Carl Woese’s use of 16S in the 1970s revealed many uncultured bacteria and showed that <1% of environmental microbes were captured by cultivation. Shotgun metagenomics builds on this by sequencing those uncultured microbes’ entire DNA, yielding insights into their metabolic capabilities.

Strengths, Limitations, and Use Cases

• Amplicon sequencing – strengths: It is cost-effective and sensitive for surveying community composition across many samples. Because many 16S sequences can be multiplexed in one sequencing run, it’s well-suited for comparative studies (e.g. microbiomes of different patients or soils). Databases of 16S sequences allow taxonomic identification of reads. Amplicon methods are relatively straightforward with established pipelines (QIIME, Mothur, etc.).
• Amplicon – limitations: PCR bias can skew relative abundances (primer mismatches may under-amplify some taxa). It provides limited taxonomic resolution – 16S amplicons (~150–300 bp variable regions or even full ~1500 bp gene) often distinguish genus-level, but may not resolve closely related species or strains. It also misses functional genes and organisms lacking the targeted marker. For example, viruses are not detected by 16S (no universal gene), so viral diversity is invisible to amplicon surveys.
• Amplicon use cases: Microbiome profiling where the goal is to compare community structure (e.g. gut microbiota shifts between health and disease) or to screen many samples. It’s especially common for bacterial communities via 16S, or fungal via ITS. Results are often reported as relative abundance of taxa.
• Shotgun metagenomics – strengths: Captures comprehensive genetic information, including bacteria, archaea, fungi, viruses, and other DNA in the sample. Yields data on functional potential – genes for enzymes, metabolism, antibiotic resistance, etc., can be identified. It enables assembly of genomes or MAGs (Metagenome-Assembled Genomes) for dominant community members, allowing discovery of novel species and functions. Shotgun data can also quantify relative gene abundance and explore strain-level variation.
• Shotgun – limitations: Higher cost and sequencing effort per sample (since it doesn’t target a small region, much more sequencing is needed) – for example, clinical shotgun metagenomics tests can cost on the order of \$1000 per sample as of 2025. Data analysis is complex: requires robust bioinformatics for assembly, binning, and annotation. Short reads may assemble poorly for low-abundance organisms, and host DNA (in human-associated samples) can overwhelm sequencing. Deeper sequencing is often required to detect rare taxa compared to targeted amplicon approaches.
• Shotgun use cases: When functional insights are needed (e.g. analyzing the metabolic pathways in gut microbiome, or detecting microbial genes for bioremediation in soil). Also essential for virome analysis (since viruses lack a single marker gene) and for recovering genomes of uncultured microbes. Many large projects (Human Microbiome Project phase 2, Earth Microbiome Project) employ shotgun sequencing to catalog not just who’s there, but what they can do.